JPH08196614A - Locally absorptive hemostatic agent - Google Patents

Abstract

PURPOSE: To provide a collagen-made locally absorptive hemostatic agent that has a sufficient and stable hemostatic function and that is safely and quickly decomposed and absorbed in a living body after use, and further that is excellent in operability and inexpensive. CONSTITUTION: A locally absorptive hemostatic agent is the fibers of atherocollagen that is produced by reproducing alkali soluble collagen, and the water-solubility thereof is 30 to 60%, and it is formed by working the fibers whose HR within 10 minutes is 100%, in a desired shape.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a locally absorbable hemostatic material comprising collagen fibers used in the field of surgery, and particularly to capillary hemorrhage from a solid organ or hemorrhage from a blood vessel anastomosis site. The present invention relates to a locally absorbable hemostatic material that can be quickly and effectively applied.

[0002]

2. Description of the Related Art As a hemostatic method during surgery, a compression method,
There are ligation method, electrocoagulation method, and application of physiologically active substances such as thrombin and fibrin glue. Ligation or electrocoagulation is generally used for arterial bleeding with a clear bleeding point, and if venous bleeding, compression is sufficient and hemostasis is easy. However, these hemostatic methods may not be effective for capillary bleeding from parenchymal organs or bleeding from vascular anastomosis, and it is especially difficult to stop bleeding in cases of hepatic failure or cardiovascular surgery. Cause In such a case, a locally absorbable hemostatic material that accelerates the blood coagulation reaction just by contacting the bleeding surface, quickly forms a thrombus, and prevents bleeding, not only shortens the operation time but also causes re-bleeding after surgery. It is also effective because it also contributes to safe postoperative management. For this purpose, various local hemostatic agents using oxidized cellulose as a material have been developed and clinically applied, but these local hemostatic agents have the advantage of being inexpensive and excellent in operability. Since it is not a material of origin, it has the drawback of being slowly absorbed in the body and inducing a strong hemolytic reaction and a foreign body reaction.In recent years, it has a low antigenicity and is safely absorbed by the body to minimize allergic reaction and foreign body reaction. The local hemostatic agent using collagen, which is a biologically-derived protein that can be retained in the body, has its own physiological activity (adhesive aggregation of platelets, and platelets released from the aggregated platelets II.
The action of factor I to promote blood coagulation and form thrombus)
Therefore, since it has a high hemostatic effect, it has been extensively applied clinically.

Collagen local hemostatic agents currently in practical use include flakes made of collagen fine fibers and flat plates made of collagen sponge. -Since telopeptide, which is the antigenic determinant of gen, remains and shows antigenicity in vivo, it must be removed after use, and since it is flaky, it is washed away by blood and scattered, so a hemostatic effect can not be expected so much However, there is a problem in terms of operation that it is easily charged with static electricity and easily adheres to hands and tweezers during use.On the other hand, the latter is made of atelocollagen without telopeptide, but is flat. Therefore, the adhesion to the wound surface with a complicated shape is not sufficient, and pressure hemostasis cannot be performed, so the same hemostatic effect as the former cannot be expected.

As a solution to the above-mentioned drawbacks, a cotton-like local hemostatic material comprising atelocollagen has been announced. For example, pig-skin-derived atelocollagen is spun and dried into a cotton shape (Shimizu et al., Artificial organ 19 (3), 1
235 (1990)) or spun collagen treated with a cross-linking agent, washed and freeze-dried to give a cotton-like shape and fine cracks on the surface (Japanese Patent Laid-Open No. 4-61862). However, the former uses soluble atelocollagen as it is, and therefore, when used for hemostasis, it may absorb blood and significantly reduce the strength of the fiber to block blood flow. It is difficult to expect a sufficient hemostatic effect. On the other hand, in the latter, the hemostatic action of collagen is impaired because the crosslinking treatment with a crosslinking agent is performed prior to freeze-drying, and in order to completely remove the unreacted crosslinking agent from the viewpoint of safety. There was a problem that a cleaning operation was required.

In order to solve these problems, solubilization treatment with a proteolytic enzyme or solubilization treatment with an alkali is carried out, and at the same time as solubilization, an operation of removing telopeptide which is an antigenic determinant of collagen is carried out. Colla
A cotton-like material composed of atelocollagen fibers obtained by regenerating gen, in which at least a part of the collagen molecules constituting the fiber is crosslinked by heat (Japanese Patent Laid-Open No. 6-19794).
No. 6) has been proposed, but it has been solubilized by a proteolytic enzyme (hereinafter, this solubilization is referred to as "enzyme solubilization (treatment)" and cotton produced through the enzyme solubilization treatment. The hemostatic material is referred to as "enzyme-solubilized (treated) product", which has extremely excellent hemostatic performance, but the production cost is high due to the use of enzyme. On the other hand, solubilization treatment with alkali was performed. (Hereinafter, this solubilization treatment is called "alkali solubilization (treatment)", and the cotton-like hemostatic material made through alkali solubilization treatment is called "alkali solubilization (treatment) product".
Each of these) can be processed in a large amount and the resulting collagen solution has a low viscosity and is excellent in processability such as spinning, but the hemostatic performance is not as high as that of the enzyme-solubilized (treated) product, and the stability is not high. poor.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a hemostatic material which solves the problems of the above-mentioned conventional collagen hemostatic materials, that is, it has a sufficient and stable hemostatic ability and is safe and prompt in vivo after use. It is an object of the present invention to provide a collagen local hemostatic material which is decomposed and absorbed by the method and has good operability and is inexpensive.

[0007]

The present inventors have arrived at the present invention as a result of repeated studies to achieve such an object. That is, the present invention is a fiber of atelocollagen regenerated from alkali-solubilized collagen, which has a water solubility of 30 to 60% and an HR within 100 minutes of 100%.

In the present invention, the alkali-solubilized collagen refers to collagen which has been subjected to a solubilization treatment with an alkali to remove telopeptide, which is an antigenic determinant of collagen, at the same time as solubilization. is there. The origin of the collagen used as the raw material of the present invention is not particularly limited, but in general, mammals (for example, human, bovine, porcine,
Collagen obtained from skin, bone, cartilage, tendon, organ, etc. of rabbits, sheep, mice, etc. is used. Also, birds,
A collagen-like protein obtained from fish or the like can also be used.

In the present invention, the water solubility means that the fiber or the locally absorbable hemostatic material comprising the fiber is added to warm water at 65 ° C.
The solubility of the fiber or the locally absorbable hemostatic material comprising the fiber when soaked for a minute, which is an index adopted because this index is in a proportional relationship with the crosslinking degree of the collagen molecule constituting the fiber. On the other hand, HR is an index showing the degree of hemostasis when the fiber is applied as a certain form of locally absorbable hemostatic material (the fiber itself has hemostatic ability due to its physiologically active characteristics, As described below, this index was used because the actual hemostatic ability cannot be properly evaluated unless the fiber is applied as a locally absorbable hemostatic material). The specific method of obtaining the HR is as follows. 50 mg of the fiber used as a locally absorbable hemostatic material in a desired form is applied to a target site of hemostasis and manually pressed for 30 seconds. After that, at a 1 minute interval, a gauze is applied from above the hemostatic material for 30 seconds to measure BD (the average value of the major axis and the minor axis of the circle drawn by the sucked blood). The BD is measured until it becomes a trace (complete hemostasis at this point) or after 10 minutes. HR is calculated according to the following formula. Where BD _{t = 0} is BD
The initial value of BD _{t = t} is BD after t minutes. HR (%) = (1- (BD _{t = t} / BD _{t = 0} )) × 100

Hemostasis by the collagen hemostatic material is carried out by first physically inhibiting the blood flowing out and then exerting the hemostatic action of collagen itself. Therefore,
The form of the hemostatic material may be, for example, cotton-like, flat-plate-like, woven / knitted fabric, non-woven fabric, etc. as long as the above-mentioned action is not impaired. The cotton-like locally absorbable hemostatic material swells when it contains blood, and the entire volume increases. As a result, it is preferable because it exerts a hemostatic effect on the tissue surrounding the application site.

The compressive hemostatic effect is exhibited only when the hemostatic material has a certain level of strength. However, the locally absorbable hemostatic material obtained by only alkali solubilization is subjected to alkali solubilization treatment. When this happens, the amino acid residue of collagen is modified, and the isoelectric point becomes low, and most of the fibers that make up the hemostatic material dissolve in the body fluid due to water in the body fluid and lose the fiber structure. Therefore, the strength decreases as the swelling occurs.

Therefore, in the present invention, first, at least a part of the collagen molecule constituting the fiber is crosslinked by heat,
We decided to give it appropriate swelling and strength as a hemostatic material.

The factors that determine the degree of crosslinking are temperature and time. In the low temperature range of 90 to 120 ° C., the crosslinking slowly enters until about 24 to 48 hours. At 130-140 ° C., crosslinking reaches a plateau within a few hours. Crosslinking occurs rapidly in a high temperature range of 150 ° C or higher. On the other hand, the relationship between the degree of crosslinking and the hemostatic ability is as follows. When the amount of cross-linking is small (water solubility is 40% or more), the fibers constituting the hemostatic material are remarkably dissolved and local bleeding cannot be stopped. Medium degree of cross-linking (water solubility of about 30%)
Swells but the dissolution of the fiber is not so great, and if the pressure is continued for 10 minutes or more, hemostasis can be achieved. When the degree of cross-linking is high (water solubility is 20% or less), the fibers become hard, and when the blood is contained, the hemostatic material becomes a lump of solid, and at the beginning of application, hemostasis is started but after a few minutes. The whole hemostatic material is pushed up by the bleeding blood, and bleeding continues (the temperature of thermal crosslinking is 1
When the temperature exceeds 50 ° C, denaturation of collagen itself begins to occur and the physiologically active action originally possessed by collagen begins to be impaired).

As described above, if an appropriate temperature and time are selected, even an alkali-solubilized product will exhibit a hemostatic ability equivalent to that obtained by thermal crosslinking. Complete hemostasis cannot be reached within HR = 100%).

Then, next, the thermally crosslinked fiber was further irradiated with γ-rays. Cross-linking and cleavage of collagen molecules occur when γ-ray irradiation is performed, but if the irradiation conditions are selected in an appropriate region, the proportion of collagen molecules that are cut is higher than the increase in cross-linking, and the hemostatic agent is softened. This is because the water solubility increases as the temperature increases. In addition, gamma irradiation also leads to improved platelet aggregation function (due to the large number of amide groups created).

For the above-mentioned reason, the processing conditions are as follows. A point (130 ° C., 8 hours) in a rectangular coordinate system with the processing temperature on the vertical axis (or the horizontal axis) and the processing time on the horizontal axis (or the vertical axis). ), Point B (130 ° C, 10 hours), point C (145 ° C,
It is preferable to perform γ-ray irradiation after thermal crosslinking within a range surrounded by point D (145 ° C., 5 hours) for 3 hours). The point A is 130 ° C. for 8 hours, and the point B is 130 ° C., 10
Time, C point is 140 ° C, 5 hours, D point is 140 ° C,
It is more preferably within 7 hours, and the point A is 1
35 ° C, 6 hours, point B, 135 ° C, 8 hours, point C
It is particularly preferable that the temperature is 138 ° C. for 5.5 hours and the point D is 138 ° C. for 6.5 hours. If the heat treatment time is short, the heat treatment is more likely to be uneven, while
This is because if it is too long, the manufacturing cost will increase. Incidentally, the heat treatment is, in the sense of ensuring continuity with the γ-ray irradiation treatment,
It may be performed after processing the fiber of the present invention into a desired form as a hemostatic agent. On the other hand, in the sense that uniform heat treatment is performed, it is performed after spinning before processing into a desired form as a hemostatic agent. Good. Here, the irradiation dose of γ-ray is preferably at least 2.5 Mrads. This is because the dose is required for sterilization, and the dose of the cleavage of the collagen molecule is higher than the increase of the crosslinking of the collagen molecule.

The water solubility value of the fiber of the present invention or the hemostatic material comprising the fiber, which has been mentioned as a preferred range, is 40 to 5
0% is the range of heat treatment conditions performed before the γ-ray irradiation treatment, that is, the point A is 130 ° C. for 8 hours, and the point B is 1
The conditions correspond to 30 ° C., 10 hours, point C, 140 ° C., 5 hours, and point D, 140 ° C., 7 hours.

Another physical property that should be possessed as a locally absorbable hemostatic material is that the diameter of the constituent fibers (thickness increases strength,
If it is too thick, the fibers become rigid and it becomes difficult to process the shape, so 10 to 70 μm is preferable, and 2 is preferable.
0 to 50 μm), the length of the constituent fibers (because the fibers are obtained by regenerating soluble collagen, any length can be obtained, but if the length is too long, it is sufficient for forming a cotton-like shape. It becomes difficult to disperse, and if it is too short, the fibers do not easily get entangled with each other and are scattered at the time of application of the hemostatic material, and the strength against blood pressure cannot be obtained. Therefore, 3 to 70 mm is preferable, and 20 to 50 mm is preferable. , And the specific volume of cotton-like material (ease of shape processing when applied,
In other words, the shape of the hemostatic material can be changed according to the wound site with various shapes, and the hemostatic effect can be maximized by evenly adhering the hemostatic material. The higher the better, but the fibers are appropriately entangled with each other. For that reason, there is a limit naturally, and if it is too high, it will be difficult to completely stop the bleeding because fibers will be scattered or gaps will be created during application, so 20 to 80 cm ³ / g is preferable, and preferably 40
˜70 cm ³ / g). here,
The specific volume is obtained as follows (hereinafter, described with reference to the cotton fiber of collagen fiber,
The same thing is measured for lumps). The obtained cotton-like material of collagen fibers was put in a standard state (20
Approximately 1 g is taken under (° C, 65% RH) and accurately weighed (measured weight: Wg). Under standard conditions, it is uniformly filled in a transparent plastic cylinder with an inner diameter of 35 mm. Next, a flat disk with a diameter of 30 mm and a weight of 5.0 g
It is placed on the cotton-like material prepared in step 1, and a weight of 50 g is placed on the cotton-like material for 30 seconds, and then the weight is removed and left for 30 seconds. This operation is repeated 3 times, and then the height of the filled cotton-like material is measured at 3 locations in the circumferential direction (the average value is Hmm.
And). The specific volume was calculated for three samples according to the following formula,
The average value is adopted. Specific volume (cm ³ / g) = ((35/20) ² × π × H /
10) / W

[0019]

EXAMPLES The present invention will be described in detail below by taking a cotton-like material, which is one form of the locally absorbable hemostatic material of the present invention, as an example. Example 1 The insoluble collagen obtained from fresh cowhide was treated with an alkali with a mixed solution of sodium sulfate, sodium hydroxide and monomethylamine (the same applies hereinafter) to digest telopeptide and obtain a solubilized atelocollagen solution. Next, this was dissolved in a hydrochloric acid aqueous solution adjusted to pH 3 (collagen concentration: 6%), and atelocollagen fibers were obtained by a wet spinning method using 20% ammonium sulfate as a coagulating liquid. The obtained atelocollagen fiber is desalted with methanol, dehydrated, dried, and then cut into a length of 50 mm,
The fibers were dispersed by air blow (30 m / s wind speed for 10 minutes), then heat-treated at 140 ° C. for 5 hours under reduced pressure, and further irradiated with 25 kGy of γ-ray to obtain a collagen fiber cotton. . The physical properties of the obtained cotton-like material are: water solubility: 49%, fiber length: 20 to 50 mm, fiber diameter:
It was 20 to 50 μm and the specific volume was 60 cm ³ / g.

Examples 2 to 6 Cotton fibers of collagen fibers were obtained in the same manner as in Example 1 except that the heat treatment was carried out under the conditions shown in Table 1. The physical properties of the obtained cotton-like material were the same as those in Example 1 except that the water solubility was the value shown in Table 1, respectively.

Comparative Examples 1 to 16 Cotton fibers of collagen fiber were prepared in the same manner as in Example 1 except that the heat treatment was carried out under the conditions shown in Table 1 and the γ-ray irradiation after the heat treatment was not performed. Got The physical properties of the obtained cotton-like material were the same as those in Example 1 except that the water solubility was the value shown in Table 1, respectively.

Comparative Example 17 A cotton fiber material of collagen fiber was obtained in the same manner as in Example 1 except that the heat treatment was carried out under the conditions shown in Table 1. The physical properties of the obtained cotton-like material were the same as those in Example 1 except that the water solubility was the value shown in Table 1, respectively.

Reference Example A cotton-like material of collagen fibers was obtained in the same manner as in Example 1 except that the enzyme solubilization treatment with pepsin was applied instead of the alkali solubilization treatment. The physical properties of the obtained cotton-like material are
Same as Example 1 except that the water solubility was 22%.

[Evaluation of Hemostatic Effect] A hybrid adult dog was subjected to laparotomy under general anesthesia to expose the spleen, and 100 u / kg of heparin was intravenously administered once, and then the capsule of the spleen was 1 cm × 1 cm with a scalpel.
Largely resected. Next, apply dry gauze to the wound surface for 30 seconds,
BD (Breeding Degree) was measured. The first BD is 40
Bleeding wounds having a size of not less than mm and not more than 40 mm were not used in the experiment. One minute after the coating was peeled off, 50 mg of each collagen cotton-like material obtained in each of the above Examples, Comparative Examples and Reference Example was applied to the wound surface and manually pressed for 30 seconds. After that, the BD was measured by applying a gauze for 30 seconds on the cotton at intervals of 1 minute until the blood adhering to the gauze became a trace (at this point, complete hemostasis), or 10 minutes. BD was measured until later, and the hemostasis rate: HR was calculated according to the following formula. here,
BD _{t = 0} is the initial value of BD, and BD _{t = t} is the BD after t minutes. HR (%) = (1− (BD _{t = t} / BD _{t = 0} )) × 100 Table 1 shows data when 4 cases were performed.

[0025]

[Table 1]

As shown in Table 1, it is clear that the hemostatic collagen cotton-like material according to the present invention has the same hemostatic ability as that of the enzyme-solubilized product (reference example) (those subjected to alkali solubilization alone). In the comparative example, complete hemostasis was not obtained within the predetermined time).

Further, in order to confirm the stability of hemostatic ability,
A similar experiment was conducted using the hemostatic collagen cotton-like material of Example 1, Comparative Example 7 and Reference Example (the number of experiments is the same as Example 1).
And 8 times for the cotton-like material of Comparative Example 7, and 5 times for that of the reference example). The results are shown in FIG. 1, and it was found that the hemostatic collagen cotton-like material according to the present invention obtained complete hemostasis within a predetermined time in all experiments and had a stable hemostasis ability. On the other hand, in the case of performing only alkali solubilization (Comparative Example 7), complete hemostasis was not obtained in 3 out of 8 times, and even in 5 cases in which complete hemostasis was obtained, compared to the product of the present invention, There was an impression that the adhesiveness was poor.

[In-vivo absorption and tissue reaction] Five hybrid dogs were subjected to laparotomy under general anesthesia to expose the spleen, and heparin was not administered, and the spleen capsule was excised with a scalpel to a size of 1 cm x 1 cm. did. Next, 50 mg of each of the hemostatic collagen cotton wool of Example 1, Comparative Example 7 and Reference Example was attached to the wound surface, and manually pressed until the hemostasis was completed, and after the completion of hemostasis was confirmed, the abdomen was closed. . Two animals were sacrificed one week after the operation and three animals were sacrificed three weeks later, and the degree of absorption of each cotton-like material, tissue reaction, etc. were examined histopathologically.

In all 5 animals, there was no significant change in general condition from the time of operation until sacrifice, and no signs of hemorrhage, peritonitis, etc. were observed when relapsing. One week later, histologically, all three types were significantly changed from those immediately after the operation, no fibrous structure was observed, and cell infiltration into collagen was small. After 3 weeks, only the white tissue was observed in all three types, and the hemostatic agent could not be visually confirmed. Histologically, in the product of the present invention and the enzyme-solubilized product (reference example), almost all collagen was absorbed and almost no cell infiltration was observed, and a thin fibrous tissue covered the spleen. What was only solubilized (Comparative Example 7)
In this case, a part of collagen remained, and infiltration of cells such as neutrophils and macrophages was observed around it. It was confirmed that the product of the present invention is a local hemostatic agent which is rapidly absorbed into the living body and has little tissue reaction, and is advantageous also in this respect.

[0030]

EFFECTS OF THE INVENTION The collagen fiber according to the present invention has a sufficient and stable hemostatic ability, is decomposed and absorbed safely and promptly in vivo after use, and has the same characteristics as an enzyme-solubilized product, which has good operability. The locally absorbable hemostatic material can be provided at a lower cost than the enzyme-solubilized product.

[Brief description of drawings]

FIG. 1 is a diagram showing a result of confirmation of stability of hemostatic ability of a hemostatic material of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuya Kiyoya 34-35 Tanakagami Furukawa-cho, Sakyo-ku, Kyoto City, Maison Takano 307 (72) Inventor Masami Teramachi 3-8, Fukakusa-Nishiura-cho, Fushimi-ku, Kyoto-shi, Kyoto Prefecture 9 Shah Fukakusa 1st 40B (72) Inventor Norihito Okumura 5-3 1-3 Kojidai, Nishi-ku, Kobe-shi, Hyogo Prefecture Avvenir Nishi-Kobe 208

Claims (9)

[Claims] 1. A fiber of atelocollagen regenerated from alkali-solubilized collagen, which has a water solubility of 30 to 60.
%, And HR within 10 minutes is 100%. 2. The water solubility is 40 to 50%.
The fiber according to claim 1. 3. A cotton-like material comprising the fiber according to claim 1 or 2. 4. The cotton-like material according to claim 3, which has a specific volume of 20 to 80 cm ³ / g. 5. A method for producing fibers of atelocollagen by regenerating alkali-solubilized collagen, wherein at least a part of collagen molecules constituting the fibers is crosslinked by heat, and γ-ray irradiation is further applied. How to characterize. 6. The cross-linking by heat has a treatment temperature on the vertical axis (or the horizontal axis) and a treatment time on the horizontal axis (or the vertical axis) in an orthogonal coordinate system, at point A (130 ° C., 8 hours), B Point (13
The method according to claim 5, wherein the method is performed within a range surrounded by a point C (145 ° C, 3 hours), a point C (145 ° C, 3 hours), and a point D (145 ° C, 5 hours). 7. The point A is 130 ° C. for 8 hours, the point B is 130 ° C. for 10 hours, and the point C is 140 ° C. for 5 hours,
The method according to claim 5, wherein the point D is 140 ° C. and 7 hours. 8. The point A is 135 ° C. for 6 hours, the point B is 135 ° C. for 8 hours, the point C is 138 ° C. for 5.5 hours, and the point D is 138 ° C. for 6.5 hours. The method according to claim 6, wherein 9. The method according to claim 5, wherein the gamma irradiation is performed at a dose of at least 25 kGy.